TY - JOUR
T1 - Time-lapse imaging of microRNA activity reveals the kinetics of microRNA activation in single living cells
AU - Ando, Hideaki
AU - Hirose, Matsumi
AU - Kurosawa, Gen
AU - Impey, Soren
AU - Mikoshiba, Katsuhiko
N1 - Publisher Copyright:
© 2017 The Author(s).
PY - 2017/12/1
Y1 - 2017/12/1
N2 - MicroRNAs (miRNAs) are small, non-coding RNAs that play critical roles in the post-transcriptional regulation of gene expression. Although the molecular mechanisms of the biogenesis and activation of miRNA have been extensively studied, the details of their kinetics within individual living cells remain largely unknown. We developed a novel method for time-lapse imaging of the rapid dynamics of miRNA activity in living cells using destabilized fluorescent proteins (dsFPs). Real-time monitoring of dsFP-based miRNA sensors revealed the duration necessary for miRNA biogenesis to occur, from primary miRNA transcription to mature miRNA activation, at single-cell resolution. Mathematical modeling, which included the decay kinetics of the fluorescence of the miRNA sensors, demonstrated that miRNAs induce translational repression depending on their complementarity with targets. We also developed a dual-color imaging system, and demonstrated that miR-9-5p and miR-9-3p were produced and activated from a common hairpin precursor with similar kinetics, in single cells. Furthermore, a dsFP-based miR-132 sensor revealed the rapid kinetics of miR-132 activation in cortical neurons under physiological conditions. The timescale of miRNA biogenesis and activation is much shorter than the median half-lives of the proteome, suggesting that the degradation rates of miRNA target proteins are the dominant rate-limiting factors for miRNA-mediated gene silencing.
AB - MicroRNAs (miRNAs) are small, non-coding RNAs that play critical roles in the post-transcriptional regulation of gene expression. Although the molecular mechanisms of the biogenesis and activation of miRNA have been extensively studied, the details of their kinetics within individual living cells remain largely unknown. We developed a novel method for time-lapse imaging of the rapid dynamics of miRNA activity in living cells using destabilized fluorescent proteins (dsFPs). Real-time monitoring of dsFP-based miRNA sensors revealed the duration necessary for miRNA biogenesis to occur, from primary miRNA transcription to mature miRNA activation, at single-cell resolution. Mathematical modeling, which included the decay kinetics of the fluorescence of the miRNA sensors, demonstrated that miRNAs induce translational repression depending on their complementarity with targets. We also developed a dual-color imaging system, and demonstrated that miR-9-5p and miR-9-3p were produced and activated from a common hairpin precursor with similar kinetics, in single cells. Furthermore, a dsFP-based miR-132 sensor revealed the rapid kinetics of miR-132 activation in cortical neurons under physiological conditions. The timescale of miRNA biogenesis and activation is much shorter than the median half-lives of the proteome, suggesting that the degradation rates of miRNA target proteins are the dominant rate-limiting factors for miRNA-mediated gene silencing.
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U2 - 10.1038/s41598-017-12879-2
DO - 10.1038/s41598-017-12879-2
M3 - Article
C2 - 28974737
AN - SCOPUS:85030570370
SN - 2045-2322
VL - 7
JO - Scientific Reports
JF - Scientific Reports
IS - 1
M1 - 12642
ER -